Creative Bioreactor Designs, Inc

Capturing the stem cell paracrine effect using heparin-presenting nanofibres to treat cardiovascular diseases

9th March 2010

The mechanism for stem cell-mediated improvement following acute myocardial infarction has been actively debated. We support hypotheses that the stem cell effect is primarily paracrine factor-linked. We used a heparin-presenting injectable nanofibre network to bind and deliver paracrine factors derived from hypoxic conditioned stem cell media to mimic this stem cell paracrine effect. Our self-assembling peptide nanofibres presenting heparin were capable of binding paracrine factors from a medium phase. When these factor-loaded materials were injected into the heart following coronary artery ligation in a mouse ischaemia-reperfusion model of acute myocardial infarction, we found significant preservation of haemodynamic function. Through media manipulation, we were able to determine that cru...

A composite material model for improved bone formation

7th March 2010

In this study we attempted to generate a composite bone substitute maximizing the available osteoconductive surface for cell adhesion and activity. Highly porous scaffolds were prepared through a particulate leaching method, combining poly-[epsiv]-caprolactone (PCL) and hydroxyapatite (HA) particles, previously coated with a sucrose layer, to minimize their embedding by the polymer solution. Composite performances were evaluated both in vitro and in vivo. In PCL-sucrose-coated HA samples, the HA particles were almost completely exposed and physically distinct from the polymer mesh, while uncoated control samples showed ceramic granules massively covered by the polymer. In vivo results revealed a significant extent of bone deposition around all sucrose-coated HA granules, while only parts o...

Biocompatibility of synthetic poly(ester urethane)/polyhedral oligomeric silsesquioxane matrices with embryonic stem cell proliferation and differentiation

7th March 2010

In this study, we present detailed microscopic analysis of the PEU/POSS matrix and demonstrate its biocompatibility with cell culture. The PEU/POSS polymer forms a continuous porous matrix with open pores and interconnected grooves. From SEM image analysis, it is calculated that there are about 950 pores/mm2 of the matrix area with pore diameter size in the range 1-15 µm. The area occupied by the pores represents approximately 7.6% of the matrix area. Using mouse embryonic stem cells (ESCs), we demonstrate that the PEU/POSS matrix provides excellent support for cell proliferation and differentiation. Under the cell culture condition optimized to maintain self-renewal, ESCs grown on a PEU/POSS matrix exhibit undifferentiated morphology, express pluripotency markers and have a similar growt...

Action of recombinant human BMP-2 on fracture healing in rabbits is dependent on the mechanical environment

7th March 2010

The utility of recombinant human bone morphogenetic protein-2 (rhBMP-2) in inducing bone formation in fractures of bone is well known. However, the influence of the mechanical environment on the actions of rhBMP-2 on fracture healing is not clear. An experimental model of fractures of the tibia in rabbits was developed and utilized to investigate the role of mechanical environment on rhBMP-2 action. A 1 mm osteotomy gap was stabilized by either a low- or high-stiffness fixator (LSF or HSF, respectively), and local treatment with rhBMP-2 in an absorbable collagen sponge (ACS) was evaluated. The results of the investigation were analysed by both histomorphometry and biomechanics. The LSF caused an increase in mineralized periosteal callus compared to HSF, the rhBMP-2 in ACS accelerated fract...

Continuous supply of TGF[beta]3 via adenoviral vector promotes type I collagen and viability of fibroblasts in alginate hydrogel

3rd March 2010

In recent years, transforming growth factor-[beta]3 (TGF[beta]3) has interested more and more researchers with its competence in engineered histogenesis. In the present study we employed recombinant adenoviral vectors to deliver the constitutively active TGF[beta]3 gene to human dermal fibroblasts, which could maintain the continuous secretion of TGF[beta]3 from the cells. The expression of type I collagen in the Ad-TGF[beta]3 group increased significantly in comparison with other three groups: Neg (cells without treatment of the adenovirus), Ad-null (cells with treatment of the adenovirus, without the inserted gene) and Ad-shRNA (cells with treatment of the adenovirus encoding shRNA specific for type I collagen). Additionally, we demonstrated that TGF[beta]3 enhanced the expression of Sma...

Isolation of pig bone marrow mesenchymal stem cells suitable for one-step procedures in chondrogenic regeneration

28th January 2010

Large animals such as pigs are good models for skeletal tissue engineering, since they provide physical forces similar to those of humans. Porcine bone marrow mesenchymal stem cells (BMSCs) have shown regenerative capacity similar to those of human BMSCs and can therefore be preclinically applied in settings corresponding to autologous transplantation in patients. Aiming at a one-step procedure for cartilage regeneration with autologous BMSCs, three straightforward isolation methods for BMSCs of Göttingen minipigs were compared. For this purpose, the BMSC fraction was enriched by red blood cell (RBC) lysis, dextran sedimentation or density gradient centrifugation. Isolated BMSCs were evaluated with regard to cell yield, proliferation capacity, phenotype and ability to differentiate to the...

Surface modification of a biodegradable composite by UV laser ablation: in vitro biological performance

27th January 2010

Melt blends of chitosan and biodegradable aliphatic polyester have been physically and biologically studied, presenting great potential for biomedical applications. Structurally, poly(butylene succinate)-chitosan (PBS/Cht) composite scaffolds are covered by a thin PBS layer, preventing the desired interaction of cells/tissues with the chitosan particules. In the present work, a selective and controlled ablation of this skin layer was induced by UV laser processing. X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS) data demonstrated an increment of chitosan components and others resulting from the laser ablation process. The biological activity (i.e. cell viability and proliferation) on the inner regions of the composite scaffolds is not si...

Fine-tuning scaffolds for tissue regeneration: effects of formic acid processing on tissue reaction to silk fibroin

27th January 2010

This study indicates that by modifying the FA treatment time, the tissue reaction to SF scaffolds can be tailored for different tissue-engineering applications. The tunability and biocompatibility of SF make it an attractive scaffold for exploration in regenerative medicine and clinical tissue engineering. Copyright © 2010 John Wiley & Sons, Ltd. (Source: Journal of Tissue Engineering and Regenerative Medicine)

Human progenitor-derived endothelial cells vs. venous endothelial cells for vascular tissue engineering: an in vitro study

27th January 2010

The isolation of endothelial progenitor cells from human peripheral blood generates a great hope in vascular tissue engineering because of particular benefit when compared with mature endothelial cells. We explored the capability of progenitor-derived endothelial cells (PDECs) to line fibrin and collagen scaffolds in comparison with human saphenous and umbilical cord vein endothelial cells (HSVECs and HUVECs): (a) in a static situation, allowing definition of the optimal cell culture conditions with different media and cell-seeding densities to check cell behaviour; (b) under shear stress conditions (flow chambers or tubular vascular constructs), allowing investigation of cell response and mRNA expression on both substrates by oligonucleotide microarray analysis and quantitative real-time ...

Quantitative imaging of cartilage and bone for functional assessment of gene therapy approaches in experimental arthritis

25th January 2010

Anti-inflammatory gene therapy can inhibit inflammation driven by TNF[alpha] in experimental models of rheumatoid arthritis. However, assessment of the therapeutic effect on cartilage and bone quality is either missing or unsatisfactory. A multimodal imaging approach, using confocal laser scanning microscopy (CLSM) and micro-computed tomography (µCT), was used for gathering 3D quantitative image data on diseased and treated murine joints. As proof of concept, the efficacy of anti-TNF-based gene therapy was assessed, comparing imaging techniques with classical investigations. SCID mice knees were injected with human synoviocytes overexpressing TNF[alpha]. Two days later, electric pulse-mediated DNA transfer was performed after injection of the pGTRTT-plasmid containing a dimeric soluble-TN...

Porous polylactide/[beta]-tricalcium phosphate composite scaffolds for tissue engineering applications

17th January 2010

This study indicated that no considerable change was noticed in the structure of the scaffolds when the [beta]-TCP filler was added. Also, the amount of [beta]-TCP did not affect the pore size or the pore distribution in the scaffolds. We observed that the fabrication method improved the thermal stability of the samples. Our results suggest that, from the structural point of view, these scaffolds could have potential for the treatment of osteochondral defects in tissue engineering applications. The porous bottom surface of the scaffold and the increased osteogenic differentiation potential achieved with [beta]-TCP particles may encourage the growth of bone cells. In addition, the dense surface skin of the scaffold may inhibit the ingrowth of osteoblasts and bone tissue, while simultaneousl...

Fabrication of scaffold-free contractile skeletal muscle tissue using magnetite-incorporated myogenic C2C12 cells

17th January 2010

We have fabricated a functional skeletal muscle tissue using magnetite-incorporated myogenic cell line C2C12 and a magnetic field. Magnetite-incorporated C2C12 cells were patterned linearly on a monolayer of fibroblast NIH3T3 cells, using a magnetic field concentrator. After induction of differentiation, the C2C12 cells fused and formed multi-nucleated myotubes. The 3T3 layer became detached in a sheet-like manner after cultivation in differentiation medium for 5-8 days. When two separate collagen films were placed on a culture dish as tendon structures, a cylindrical construct was formed. Histological observation of the fabricated cylindrical tissue revealed the presence of multinucleate cells within it. Immunofluorescence staining of the construct showed the presence of sarcomere structu...

Synthetic poly(amino acid) hydrogels with incorporated cell-adhesion peptides for tissue engineering

17th January 2010

Preparation of soft poly(amino acid) hydrogels containing biomimetic cell-adhesive peptides was investigated. Covalently crosslinked gels were formed by radical co-polymerization of methacryloylated macromonomer poly[N5-(2-hydroxyethyl)-L-glutamine-stat-L-alanine-stat-methacryloyllysine] with 2-hydroxyethyl methacrylate (HEMA) as minor co-monomer. Hydrogels carrying biomimetic peptides were prepared by using methacryloylated peptides, such as methacryloyl-GGGRGDSG-OH and methacryloyl-GGGYIGSR-OH, as additional monomers in the polymerization mixture. Mechanical stability and swelling in water of the hydrogels obtained for different solid:water and polypeptide:HEMA ratios were evaluated. The microporosity of gels (5-20 µm), dependent on the polyHEMA phase separation in water, was followed b...

Induced pluripotent stem cells (iPSCs): the emergence of a new champion in stem cell technology-driven biomedical applications

17th January 2010

Pluripotent stem cells possess the unique property of differentiating into all other cell types of the human body. Further, the discovery of induced pluripotent stem cells (iPSCs) in 2006 has opened up new avenues in clinical medicine. In simple language, iPSCs are nothing but somatic cells reprogrammed genetically to exhibit pluripotent characteristics. This process utilizes retroviruses/lentiviruses/adenovirus/plasmids to incorporate candidate genes into somatic cells isolated from any part of the human body. It is also possible to develop disease-specific iPSCs which are most likely to revolutionize research in respect to the pathophysiology of most debilitating diseases, as these can be mimicked ex vivo in the laboratory. These models can also be used to study the safety and efficacy o...

Scaffold-free cell sheet injection results in bone formation

17th January 2010

We previously reported a new cell transplantation method in which mesenchymal stem cells (MSCs) were cultured as cell sheets. The cultured MSC sheets showed high alkaline phosphatase (ALP) activities and osteocalcin (OC) contents. In the present study, we transplanted such sheets by injection to assess whether the injectable MSC sheets could form bone tissue at subcutaneous sites. At 4 weeks after the subcutaneous injection, the injected areas showed hard mass formation. Each mass consisted of newly formed bone, as evaluated by radiographic, histological and gene expression analyses as well as three-dimensional computed tomography (3D-CT). Histological analyses revealed extracellular bone matrix together with osteocytes and active osteoblasts. Real-time PCR analyses showed high ALP and OC ...

Laryngotracheal reconstruction with porous titanium in rabbits: are vascular carriers and mucosal grafts really necessary?

7th January 2010

Laryngotracheal reconstruction requires a supportive structure with a mucosal lining, which needs a vascular supply in order to regenerate properly. We investigated the necessity of a vascular carrier and mucosal graft when using porous titanium for laryngotracheal reconstruction. Surgical defects of the laryngotracheal complex in 22 rabbits were reconstructed with: (a) porous titanium implanted on a vascularized fascia combined with a buccal mucosal graft (first stage) before transposing to the neck area (second stage); (b) porous titanium implanted on a vascularized fascia (first stage) combined with a mucosal graft (second stage); (c) porous titanium on a pedicled fascia flap; and (d) porous titanium alone. The grafts were tolerated well. Re-epithelialization occurred in all groups. Nor...

Silk fibroin microparticles as carriers for delivery of human recombinant BMPs. Physical characterization and drug release

6th January 2010

Bone morphogenetic proteins (BMPs) are cytokines with strong ability to promote new bone formation. Herein, we report the use of silk fibroin microparticles as carriers for the delivery of BMP-2, BMP-9 or BMP-14. BMP-containing fibroin microparticles were prepared by a mild methodology using dropwise addition of ethanol, exhibiting mean diameters of 2.7 ± 0.3 µm. Encapsulation efficiencies varied between 67.9 ± 6.1 % and 97.7 ± 2.0 % depending on the type and the amount of BMP loaded. Release kinetics showed that BMP-2, BMP-9 and BMP-14 were released in two phases profile, with a burst release in the first two days followed by a slower release, for a period of 14 days. The release data were best explained by Korsmeyer's model and the Fickian model of drug diffusion. Silk fibroin microp...

Dexamethasone treatment during the expansion phase maintains stemness of bone marrow mesenchymal stem cells

6th January 2010

In conclusion, the collective data argue that low-dose Dex preserves the stemness of hMSCs during repeated passaging, as indicated by the maintenance of the stem cell phenotype, proliferative capacity and multi-lineage differentiation potential. Copyright © 2010 John Wiley & Sons, Ltd. (Source: Journal of Tissue Engineering and Regenerative Medicine)

Novel nanostructured scaffold for osteochondral regeneration: pilot study in horses

3rd January 2010

The present in vivo preliminary experiment is aimed at testing mechanical and biological behaviour of a new nano-structured composite multilayer biomimetic scaffold for the treatment of chondral and osteochondral defects. The three-dimensional biomimetic scaffold (Fin-Ceramica Faenza S.p.A., Faenza - Italy) was obtained by nucleating collagen fibrils with hydroxyapatite nanoparticles, in two configurations, bi- and tri-layered, to reproduce, respectively, chondral and osteochondral anatomy. Chondral defects (lateral condyle) and deep osteochondral defects (medial condyle) were made in the distal epiphysis of the third metacarpal bone of both forelimbs of two adult horses and treated respectively with the chondral and osteochondral grafts. Both animals were euthanised six months follow up. ...

Immobilization of fibronectin in chitosan substrates improves cell adhesion and proliferation

3rd January 2010

This study showed that Fn may be easily covalently attached onto chitosan substrates, improving the biological performance of the material. The technique could find applications in tissue-engineering strategies, as the surface modification of chitosan-based substrates could be carried out in more complex geometries, such as in scaffolds or particles. Copyright © 2009 John Wiley & Sons, Ltd. (Source: Journal of Tissue Engineering and Regenerative Medicine)

Cyclic acetal hydroxyapatite composites and endogenous osteogenic gene expression of rat marrow stromal cells

3rd January 2010

In this study, bone marrow stromal cells (BMSCs) were differentiated on cyclic acetal composites containing hydroxyapatite (HA) particles (110 or 550 nm). These composites were evaluated for their role in influencing osteogenic signalling by encapsulated BMSCs. While a number of factors exert influence on osteogenic signalling during the production of an osteogenic matrix, we hypothesize that HA particles may upregulate bone growth factor expression due to enhanced BMSC adhesion. To this end, fluorescence-activated cell sorting (FACS) analysis was performed for the evaluation of BMSC surface marker expression after culture on two-dimensional (2D) cyclic acetal/HA composites. Three-dimensional (3D) composites were then fabricated by incorporating 110 or 550 nm HA particles at 5, 10 and 50 n...